The long term goal of my research program is to determine at the molecular level a biochemical basis for the toxicity of low levels of hydrogen peroxide (H2O2) and superoxide radicals (O2-) on bacteria, particularly for fastidious pathogens. The aim of the proposed research is to continue testing the working hypothesis that Legionellae are acutely sensitive to low levels of H2O2 and O2- which form in complex culture media (including yeast extract, YEB) exposed to room light by photochemical oxidative mechanisms. The problem is not unique to Legionellae, but has explained the microaerophilic nature of Campylobacter jejuni, and may well explain the difficulties encountered in cultivation of Leptospira, Neisseria, and Treponema pallidum. Since the difficulties associated with primary isolation of Legionellae from clinical and environmental sources cannot be explained nutritionally, this study is expected to provide useful information on a fundamental mechanism and ultimately may lead to formulation of an improved primary isolation medium. This hypothesis is based on the following observations: 1) detection of photochemically produced O2- and H2O2 in YEB, 2) stability of H2O2 in YEB, 3) rapid decomposition of H2O2 by the charcoal supplement of CYE broth, 4) lack of appreciable catalase activity in L. pneumophila, and 5) partial success with replacing the charcoal supplement of YEB with combinations of superoxide dismutase, catalase, horseradish peroxidase, and arterenol. The hypothesis will be further tested by determining the tolerance of the Legionellae to various levels of O2- and H2O2 and by quantifying the levels of oxygen detoxifying enzymes (SOD, catalase, and peroxidase) in the various species of Legionella. A second aim of the proposed work will be an examination of the enzymes of the Krebs cycle and the composition and function of the respiratory chain in the oxidative metabolism. This study is expected to provide useful information on the nature of the oxygen toxicity as well as on the oxidative physiology of the Legionellae.